Project Summary The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and inflammatory bowel disease (IBD). Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. The overall specific aims of this application are: Specific Aim 1. To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier. Specific Aim 2. To elucidate the role of intracellular signaling in autophagy regulation of intestinal TJ barrier. Specific Aim 3. To delineate the role of autophagy in intestinal TJ barrier function and inflammation in animal models of IBD. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.